The present invention relates to methods of identifying candidate compounds for regulating skeletal muscle mass or function, regulating the activity or expression of a vasoactive intestinal peptide receptors (VPAC) or regulating expression of vasoactive intestinal peptide (VIP) or VIP analogs. The invention also relates to methods for the treatment of skeletal muscle atrophy or methods for inducing skeletal muscle hypertrophy utilizing VPAC receptors as the target for intervention.
Each of the VPAC receptor protein sequences included in the sequence listing, along with the corresponding Genbank accession number and animal species from which it is cloned, is shown in Table 1.
VPAC and Ligands
Vasoactive intestinal peptide (VIP) and its functionally and structurally related analogs (VIP analogs), are known to have many physiological functions including smooth muscle relaxation (bronchodilation, intestinal motility), regulation of microvascular tone (vasodepression) and permeability, regulation of mucus secretion, modulation of various inmmune functions (anti-inflammation, immune cell protection), neurological effects (memory improvement, hypnogenesis, food intake, circadian rhythm control, sexual behavior), maintenance of salivary gland function, developmental growth regulation and stimulation of hormone secretion (prolactin, growth hormone, insulin). VIP and VIP analogs mediate their effects through vasoactive intestinal peptide receptors via both neuronal (as putative neurotransmitters) and neuroendocrine pathways. There are two VPAC receptors identified to date (VPAC1 and VPAC2). The VPAC1 receptor has been cloned from human, mouse (Mus musculus), rat (Rattus norvegicus and Rattus sp.), pig (Sus scrofa), frog (Rana ridibunda), goldfish (Carassius auratus), and turkey (Meleagris gallopavo). The VPAC2 receptor has been cloned from human, mouse (Mus musculus), and rat (Rattus norvegicus).
VPAC1 and VPAC2 receptors are classified in the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor family based on sequence homology to other members of the PACAP family. Receptors in the PACAP family are further subdivided into two subclasses based on ligand affinity. The PACAP type I receptors have a much greater affinity for PACAP than for VIP, while the PACAP type II receptors have an approximately equal affinity for PACAP and VIP. Because VPAC1 and VPAC2 receptors have similar affinities for PACAP and VIP, these receptors are classified as PACAP type II receptors. Selective agonists and antagonists can differentiate VPAC1 and VPAC2 receptors from each other, both molecularly and pharmacologically, as well as from the PACAP type I receptors. These agonist and antagonists have been useful in matching biological activity to a particular VPAC receptor subclass.
VPAC1 and VPAC2 receptors both belong to the G-protein coupled receptor (GPCR) class. The specificity of coupling of VPAC1 and VPAC2 receptors to a particular G-protein, appears to depend upon the tissue examined. In tissues such as muscle, agonist activation of VPAC1 or VPAC2 receptors leads to Gxcex1s activation of adenylate cyclase. Adenylate cyclase catalyzes the formation of cAMP which in turn has multiple effects including the activation of protein kinase A, intracellular calcium release and mitogen-activated protein kinase (MAP kinase) activation. In other studies, the enhancement of intracellular inositol triphosphate synthesis after agonist activation of VPAC receptors suggests VPAC receptor coupling to either Gxcex1i or Gxcex1q.
Expression of VPAC1 and VPAC2 receptors is tissue specific and the pattern of expression of each receptor differs. In humans, the VPAC1 receptor has been shown to be expressed in brain, adipose, liver, and heart, while the VPAC2 receptor has been shown to be expressed in lung, pancreas, brain, kidney, skeletal muscle, stomach, heart, and placenta. In the rat, expression of the VPAC1 receptor has been found in the pineal gland, small intestine, liver, spleen, pancreas, lung, aorta, vas deferens and brain, while expression of the VPAC2 receptor has been shown in the stomach, intestine, skeletal muscle, spleen, pancreas, thymus, adrenal gland, heart, lung, aorta, brain, pituitary, and olfactory bulb.
Skeletal Muscle Atrophy and Hypertrophy
Skeletal muscle is a plastic tissue which readily adapts to changes in either physiological demand for work or metabolic need. Hypertrophy refers to an increase in skeletal muscle mass while skeletal muscle atrophy refers to a decrease in skeletal muscle mass. Acute skeletal muscle atrophy is traceable to a variety of causes including, but not limited to: disuse due to surgery, bed rest, or broken bones; denervation/nerve damage due to spinal cord injury, autoimmune disease, or infectious disease; glucocorticoid use for unrelated conditions; sepsis due to infection or other causes; nutrient limitation due to illness or starvation; and space travel. Skeletal muscle atrophy occurs through normal biological processes, however, in certain medical situations this normal biological process results in a debilitating level of muscle atrophy. For example, acute skeletal muscle atrophy presents a significant limitation in the rehabilitation of patients from immobilizations, including, but not limited to, those accompanying an orthopedic procedure. In such cases, the rehabilitation period required to reverse the skeletal muscle atrophy is often far longer than the period of time required to repair the original injury. Such acute disuse atrophy is a particular problem in the elderly, who may already suffer from substantial age-related deficits in muscle function and mass, because such atrophy can lead to permanent disability and premature mortality.
Skeletal muscle atrophy can also result from chronic conditions such as cancer cachexia, chronic inflammation, AIDS cachexia, chronic obstructive pulmonary disease (COPD), congestive heart failure, genetic disorders, e.g. muscular dystrophies, neurodegenerative diseases and sarcopenia (age associated muscle loss). In these chronic conditions, skeletal muscle atrophy can lead to premature loss of mobility, thereby adding to the disease related morbidity.
Little is known regarding the molecular processes which control atrophy or hypertrophy of skeletal muscle. While the initiating trigger of the skeletal muscle atrophy is different for the various atrophy initiating events, several common biochemical changes occur in the affected skeletal muscle fiber, including a decrease in protein synthesis and an increase in protein degradation and changes in both contractile and metabolic enzyme protein isozymes characteristic of a slow (highly oxidative metabolism/slow contractile protein isoforms) to fast (highly glycolytic metabolism/fast contractile protein isoforms) fiber switch. Additional changes in skeletal muscle which occur include the loss of vasculature and remodeling of the extracellular matrix. Both fast and slow twitch muscle demonstrate atrophy under the appropriate conditions, with the relative muscle loss depending on the specific atrophy stimuli or condition. Importantly, all these changes are coordinately regulated and are switched on or off depending on changes in physiological and metabolic need.
The processes by which atrophy and hypertrophy occur are conserved across mammalian species. Multiple studies have demonstrated that the same basic molecular, cellular, and physiological processes occur during atrophy in both rodents and humans. Thus, rodent models of skeletal muscle atrophy have been successfully utilized to understand and predict human atrophy responses. For example, atrophy induced by a variety of means in both rodents and humans results in similar changes in muscle anatomy, cross-sectional area, function, fiber type switching, contractile protein expression, and histology. In addition, several agents have been demonstrated to regulate skeletal muscle atrophy in both rodents and in humans. These agents include anabolic steroids, growth hormone, insulin-like growth factor I, and beta adrenergic agonists. Together, these data demonstrate that skeletal muscle atrophy results from common mechanisms in both rodents and humans.
While some agents have been shown to regulate skeletal muscle atrophy and are approved for use in humans for this indication, these agents have undesirable side effects such as hypertrophy of cardiac muscle, neoplasia, hirsutism, androgenization of females, increased morbidity and mortality, liver damage, hypoglycemia, musculoskeletal pain, increased tissue turgor, tachycardia, and edema (54th Edition of the Physicians Desk Reference, 2000). Currently, there are no highly effective and selective treatments for either acute or chronic skeletal muscle atrophy. Thus, there is a need to identify other therapeutic agents which regulate skeletal muscle atrophy.
One problem associated with identification of compounds for use in the treatment of skeletal muscle atrophy has been the lack of good screening methods for the identification of such compounds. Applicants have now found that VPAC1 and VPAC2 receptors are involved in the regulation of skeletal muscle mass or function and that agonists of VPAC1 and VPAC2 receptors are able to block skeletal muscle atrophy and/or induce hypertrophy of skeletal muscle.
The present invention relates to the use of VPAC receptors to identify candidate compounds that are potentially useful in the treatment of skeletal muscle atrophy and/or useful to induce skeletal muscle hypertrophy. In particular, the invention provides in vitro methods for identifying candidate compounds for regulating skeletal muscle mass or function. In one embodiment of the invention the method comprises: contacting a test compound with a VPAC receptor, and determining whether the test compound binds to the VPAC receptor, wherein test compounds that bind to the VPAC receptor are identified as candidate compounds for regulating skeletal muscle mass or function. In another embodiment of the invention the method comprises: contacting a test compound with a cell expressing a VPAC receptor, and determining whether the test compound activates the VPAC receptor, wherein test compounds that activate the VPAC receptor are identified as candidate compounds for regulating skeletal muscle mass or function. In yet another embodiment of the invention the method further comprises generating a list of candidate compounds.
In another aspect, the present invention relates to the use of VPAC receptors to identify candidate therapeutic compounds which regulate skeletal muscle mass or function in vivo. In particular, the invention provides a method comprising: contacting a test compound with a VPAC receptor, determining whether the test compound binds to the VPAC receptor, administering a test compound determined in the previous step to bind to the VPAC receptor, or previously known to bind to the VPAC receptor, to a non-human animal and determining whether the test compound regulates skeletal muscle mass or muscle function in the treated animal. Those test compounds that regulate skeletal muscle mass or function are identified as candidate therapeutic compounds for regulating skeletal muscle mass or function in vivo. In another embodiment of the invention the method comprises: contacting a test compound with a cell expressing VPAC receptors, determining whether the test compound activates the VPAC receptor, administering a test compound determined in the previous step to activate the VPAC receptor, or previously known to activate the VPAC receptor, to a non-human animal and determining whether the test compound regulates skeletal muscle mass or muscle function in the treated animal. Those test compounds that regulate skeletal muscle mass or function in vivo are identified as candidate therapeutic compounds for regulating skeletal muscle mass or function in vivo.
The invention further provides methods for identifying candidate compounds that prolong or augment the activation of VPAC receptors or of a VPAC receptor signal transduction pathway. These methods comprise, (i) contacting a cell which expresses functional VPAC receptors with a VPAC receptor agonist at a concentration of agonist and for a period of agonist-receptor exposure sufficient to allow desensitization of the receptor; (ii) contacting the cells with a test compound; and (iii) determining the level of activation of the VPAC receptor. In the above-described embodiment of the invention, step (ii) may be performed before or after step (i). In a particular embodiment, the present invention relates to a method of determining whether those candidate compounds that prolong or augment the agonist-induced activation of VPAC receptors or a VPAC receptor signal transduction pathway, can be used to regulate skeletal muscle mass or function in vivo by administering a candidate compound, alone or in conjunction with a VPAC receptor agonist, to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or function in the treated animal. Those candidate compounds that regulate skeletal muscle mass or function in vivo are identified as candidate therapeutic compounds for regulating skeletal muscle mass or function in vivo.
The invention further provides methods for identifying candidate compounds that increase VPAC receptor expression comprising contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a VPAC receptor gene regulatory element and detecting expression of the reporter gene. Test compounds that increase expression of the reporter gene are identified as candidate compounds for increasing VPAC receptor expression. In a particular embodiment, the present invention relates to a method of determining whether those candidate compounds which increase VPAC receptor expression can be used to regulate skeletal muscle mass or function in vivo by administering a candidate compound to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or function in the treated animals. Those candidate compounds that regulate skeletal muscle mass or function in vivo are identified as candidate therapeutic compounds for regulating skeletal muscle mass or function in vivo.
In another embodiment the invention provides for antibodies specific for VPAC receptors. In particular the invention provides for chimeric or human antibodies specific for VPAC receptors.
The invention additionally provides methods for identifying compounds that increase VIP or VIP analog expression which include the steps of contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a VIP or VIP analog gene regulatory element and detecting expression of the reporter gene. Test compounds that increase reporter gene expression are identified as candidate compounds which increase VIP or VIP analog expression. In a particular embodiment, the present invention relates to a method of determining whether those candidate compounds which increase VIP or VIP analog expression regulate skeletal muscle mass or function in vivo by administering a candidate compound to a non-human animal and determining whether the candidate compound regulates skeletal muscle mass or function in the treated animal. Those candidate compounds that regulate skeletal muscle mass or function in vivo are identified as candidate therapeutic compounds for regulating skeletal muscle mass or function in vivo.
The present invention also relates to use of a VPAC receptor as a therapeutic target to increase skeletal muscle mass or function. This use includes, the use of a VPAC receptor agonist, a compound that prolongs or augments the activation of VPAC receptors or the activation of a VPAC receptor signal transduction pathway, an expression vector encoding a functional VPAC receptor, an expression vector encoding a constitutively active VPAC receptor, a compound that increases expression of VPAC receptors, a compound that increases expression of VIP or a compound that increases expression of a VIP analog to increase skeletal muscle mass or function and/or to treat skeletal muscle atrophy. In particular, the invention provides a method for increasing skeletal muscle mass or function in a subject in which such an increase is desirable, comprising: identifying a subject in which an increase in skeletal muscle mass or function is desirable and administering to the subject a safe and effective amount of a VPAC receptor agonist, a compound that prolongs or augments the activation of VPAC receptors or the activation of a VPAC receptor signal transduction pathway, an expression vector encoding a functional VPAC receptor, an expression vector encoding a constitutively active VPAC receptor, a compound that increases expression of VPAC receptors, a compound that increases expression of VIP or a compound that increases expression of a VIP analog. In another embodiment, the invention provides methods of treating skeletal muscle atrophy, in a subject in need of such treatment, comprising administering to the subject an effective amount of a VPAC receptor agonist, a compound that prolongs or augments the activation of VPAC receptors or the activation of a VPAC receptor signal transduction pathway, an expression vector encoding a functional VPAC receptor, an expression vector encoding a constitutively active VPAC receptor, a compound that increases expression of VPAC receptors, a compound that increases expression of VIP or a compound that increases expression of VIP analog.
The present invention also relates to the use of a compound that prolongs or augmemts the activation of VPAC receptors, or of a VPAC receptor signal transduction pathway, to increase skeletal muscle mass or function and/or to treat skeletal muscle atrophy. In particular, the invention provides methods of treating skeletal muscle atrophy, in a subject in need of such treatment, comprising administering, alone or in conjunction with a VPAC receptor agonist, a safe and effective amount of a compound that prolongs or augments the activation of VPAC receptors, or of a VPAC receptor signal transduction pathway.